Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of the majority of AIDS- associated cancers. It is endemic in many areas of Africa and, due to the extraordinarily high HIV burden there, Kaposi's sarcoma is emerging as one of the most common cancers on that continent. During AIDS-induced immunosuppression, KSHV replication is no longer effectively controlled, and, together with a large latently infected population of cells, contributes to disease progression. Amplification of KSHV is dependent on its ability to exert strong control over the gene expression environment of the infected cell. A primary mechanism the virus uses to regulate gene expression is to induce widespread degradation of messenger RNA (mRNA) in the cytoplasm. This phenotype plays numerous roles in the viral lifecycle and immune evasion, and is driven by a virally encoded nuclease termed SOX. Although SOX cleaves an extremely large number of transcripts, it exhibits clear specificity for RNA Polymerase II (Pol II) transcribed RNAs and does not have significant RNA binding activity. How it recognizes and is brought to its targets remains a central unanswered question in the field and is the focus of the first Aim of this proposal. We will apply bimolecular complementation-based protein interaction screening, unbiased ribonucleoprotein complex purification, and directed functional studies to pinpoint how SOX is directed to its target mRNAs. Identification and characterization of factors that mediate SOX targeting is central to understanding how KSHV maintains control of gene expression, and may similarly provide new insight into cellular strategies to govern RNA fate. The depletion of cytosolic mRNA by SOX is presumably sensed by the cell, and thus offers a unique opportunity to reveal cellular responses to broad changes in the gene expression landscape, such as those induced by pathogens. In this regard, recent results in yeast indicate the existence of feedback mechanisms to detect and respond to altered activity of different stages of gene expression. Using gamma-herpesviruses as tools, we have now discovered an analogous pathway in mammalian cells that links mRNA degradation with transcription. Activation of this pathway, which we term `transcriptional priming', impacts the rate of nascent mRNA synthesis. Though it is likely that the normal role of this host pathway is to regulate cellular gene expression in response to stress, we hypothesize that during KSHV infection its activation by SOX instead serves to enhance Pol II transcription of viral genes. Defining how this pathway is controlled and how it impacts the viral life cycle are the goals of Aim 2. Results from these studies should provide new insight into how seemingly distant stages of gene expression are integrated, and how viral pathogens like KSHV take advantage of these cellular controls to enhance their replicative success.